The products in commerce today that are produced by Bacillus fermentations include enzymes antibiotics , and insecticides. This chapter focuses on the basic principles necessary for an understanding of growth and product production in Bacillus spp. The authors show the important link between some very basic observations and how these can be (and need to be) applied in medium development processes, choice of equipment, and elimination of bottlenecks in production. Using protease production as a model, examples show the potential value of using controlled fermentations in identifying some of the limitations of product formation in Bacillus spp. Bacillus spp. as a class are known to secrete a large number of extracellular enzymes, including several proteases , amylase, cellulase, and other degradative enzymes. Costs of exoprotein production can be estimated from material balances, molecular formulae, and growth parameters. Results of these calculations can be a key element in choosing a fermentor type and mode of operation for desired product formation. The functions included under strain development are identification and isolation of an organism exhibiting the trait (or traits) of interest; analysis and optimization of cultural conditions that allow expression of the trait; and mutagenesis, selection, and screening for isolates that hyperproduce the trait. In addition, overexpression of these proteins may improve the secretion efficiency of heterologous proteins, as has been demonstrated for signal peptidase.

Main characteristics of predominant fermentor types. Many variations on these archetypes are possible (e.g., the feed rate in an FB need not be constant). V, volume of culture; c, constant, μ, specific growth rate; x, biomass. (A) Batch fermentor (BcSc). Nutrient is present from the beginning; no feed. No steady state can be reached. (B) FB (BcSc). Nutrient is fed at a constant or variable rate; there is (usually) no flow out of the fermentor. As described in the text, growth in a carbon-limited FB can initially proceed exponentially (domain 1), after which (at least) one domain (domain 2) of linear growth occurs. No steady state can be reached. (C) CF, e.g., chemostat (BoSo). Nutrient is fed at a constant rate. Rate of inflow (F) equals rate of outflow (Fo); dilution rate (D) = F/V = c. In steady state, μ = D. (D) Combination of fermentor types. If inflow (F) equals outflow (Fo) while the filtrate flow (Ff) equals 0, this type is, again, a chemostat with V = c, μ = c, and χ = c. If Ff > 0 and Fo = 0, then D1 is a 100% RF (BcSo). V = c, μ ≠ c, χ ≠ c. Nutrient is fed at a constant rate. Rate of inflow (F) equals rate of filtrate-flow (Ff). Culture broth is continuously recycled over a cell separation unit (e.g., filter), biomass is returned to the fermentor, and filtrate is pumped off. No steady state can be reached. If Ff > 0 and Fo > 0, then D2 is a PRF (BoSo). V = c, μ = c, χ = c. As in the 100% RF, culture broth is continuously recycled over a cell separation unit. Not as in the RF, here Ff < F; part of the culture broth is pumped off at rate Fo; so, F = (Ff + Fo). In fact, a PRF is a CF type like the chemostat; here, too, D = F/V = c, but μ = Fo/V ≠ D (the Fo of a PRF is usually referred to as “bleed” [B], so μ = B/V). Nutrient is fed at a constant rate, and a steady state can be reached.

10.1128/9781555818388/fig60-1_thmb.gif

10.1128/9781555818388/fig60-1.gif

Figure 1

Main characteristics of predominant fermentor types. Many variations on these archetypes are possible (e.g., the feed rate in an FB need not be constant). V, volume of culture; c, constant, μ, specific growth rate; x, biomass. (A) Batch fermentor (BcSc). Nutrient is present from the beginning; no feed. No steady state can be reached. (B) FB (BcSc). Nutrient is fed at a constant or variable rate; there is (usually) no flow out of the fermentor. As described in the text, growth in a carbon-limited FB can initially proceed exponentially (domain 1), after which (at least) one domain (domain 2) of linear growth occurs. No steady state can be reached. (C) CF, e.g., chemostat (BoSo). Nutrient is fed at a constant rate. Rate of inflow (F) equals rate of outflow (Fo); dilution rate (D) = F/V = c. In steady state, μ = D. (D) Combination of fermentor types. If inflow (F) equals outflow (Fo) while the filtrate flow (Ff) equals 0, this type is, again, a chemostat with V = c, μ = c, and χ = c. If Ff > 0 and Fo = 0, then D1 is a 100% RF (BcSo). V = c, μ ≠ c, χ ≠ c. Nutrient is fed at a constant rate. Rate of inflow (F) equals rate of filtrate-flow (Ff). Culture broth is continuously recycled over a cell separation unit (e.g., filter), biomass is returned to the fermentor, and filtrate is pumped off. No steady state can be reached. If Ff > 0 and Fo > 0, then D2 is a PRF (BoSo). V = c, μ = c, χ = c. As in the 100% RF, culture broth is continuously recycled over a cell separation unit. Not as in the RF, here Ff < F; part of the culture broth is pumped off at rate Fo; so, F = (Ff + Fo). In fact, a PRF is a CF type like the chemostat; here, too, D = F/V = c, but μ = Fo/V ≠ D (the Fo of a PRF is usually referred to as “bleed” [B], so μ = B/V). Nutrient is fed at a constant rate, and a steady state can be reached.

Dependence of growth yield [Yxs]on reduction degree of substrate (γs) (A) and relative differences in free energies of substrate, biomasses, and exoproducts (B). (A) Growth on a substrate with γs > 4 tends to be carbon limited and energy sufficient; for γs < 4, the reverse will hold. Initially (γs < 4), the increase in free-energy content of the substrate is reflected in an increase in biomass yield (Yxs). Above a certain γs(about 4 to 4.5), ATP yield from the substrate can exceed ATP demands for biomass synthesis, and in order to sustain high metabolic rates, this surplus needs to be turned over by energy-spilling reactions (including energy-requiring exoproduct formation) (see text). (B) The free-energy content of, e.g., citrate is lower than that of biomass; bridging that gap is obtained by combusting a significant portion of the substrate to CO2 and H2O to produce energy. Concomitant synthesis of exoproduct 2 is possible; that of exoproduct 1 is very unlikely. At growth on, e.g., ethanol, a surplus of free energy that could be shunted toward synthesis of both exoproducts 1 and 2 will be available. Glucose (γs = 4) holds an intermediate position among these substrates, with a γ very close to γb(=4.2). Obviously, simultaneous consumption of two substrates with different γsvalues could also be a means of steering carbon and energy flows toward a more-desirable product formation than is possible with one substrate only. These schemes should be regarded in relation to biochemical routes. Growth on methanol, for instance, has been found to be energy limited despite the high γsof methanol. This was due to the occurrence of CO2 fixation via the costly Calvin cycle (189). For that type of growth, the substrate is actually not methanol but methanol + CO2, with a lower γsthan for methanol, (idealized drawing in panel A is based on one in reference 94; reduction degrees are as defined in reference 144).

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10.1128/9781555818388/fig60-2.gif

Figure 2

Dependence of growth yield [Yxs]on reduction degree of substrate (γs) (A) and relative differences in free energies of substrate, biomasses, and exoproducts (B). (A) Growth on a substrate with γs > 4 tends to be carbon limited and energy sufficient; for γs < 4, the reverse will hold. Initially (γs < 4), the increase in free-energy content of the substrate is reflected in an increase in biomass yield (Yxs). Above a certain γs(about 4 to 4.5), ATP yield from the substrate can exceed ATP demands for biomass synthesis, and in order to sustain high metabolic rates, this surplus needs to be turned over by energy-spilling reactions (including energy-requiring exoproduct formation) (see text). (B) The free-energy content of, e.g., citrate is lower than that of biomass; bridging that gap is obtained by combusting a significant portion of the substrate to CO2 and H2O to produce energy. Concomitant synthesis of exoproduct 2 is possible; that of exoproduct 1 is very unlikely. At growth on, e.g., ethanol, a surplus of free energy that could be shunted toward synthesis of both exoproducts 1 and 2 will be available. Glucose (γs = 4) holds an intermediate position among these substrates, with a γ very close to γb(=4.2). Obviously, simultaneous consumption of two substrates with different γsvalues could also be a means of steering carbon and energy flows toward a more-desirable product formation than is possible with one substrate only. These schemes should be regarded in relation to biochemical routes. Growth on methanol, for instance, has been found to be energy limited despite the high γsof methanol. This was due to the occurrence of CO2 fixation via the costly Calvin cycle (189). For that type of growth, the substrate is actually not methanol but methanol + CO2, with a lower γsthan for methanol, (idealized drawing in panel A is based on one in reference 94; reduction degrees are as defined in reference 144).

Theoretical exoproduct formation in batch (A) and fed batch (B) cultures. In panel A, five strains are evaluated for exoproduct formation by using batch cultures (i.e., screening stage). The same five strains are run in a carbon-limited fed-batch culture (i.e., production stage) (B). The production profiles show how the amounts of product in batch cultures at any time of sampling (without knowing the a and b values in equation 22) cannot lead to reliable predictions of exoproduct formation in fed-batch cultures. For further discussion, see text. Assumptions are that in the batch culture, growth of all five strains halted after consumption of 50 mmol of glucose because a compound other than the carbon and nitrogen source became limited. Growth in the fed-batch culture proceeded glucose limited (constant feed rate [rs] = 0.001 mol/[g · h]). Exoproduct formation is described by rp = axt + brx (21). The value of a (in grams per gram · hour) is constant; b (in grams per gram) was assumed to be described as follows: 1, b = 0.117 - 0.14μ; 2, b = 0.530 - 0.83μ; 3, b = 0.530 - 0.80μ; 4, b = 0.117 - 0.10μ; 5, b = 0.117 - 0.07μ (relations that can be obtained from, e.g., chemostat experiments). Other parameter values: V = 1 liter; Yxsm =100 g/mol; Ypsm= 60 g/mol; ms = 0.00024 mol/(g · h); x0= 0.01 g (A) or 0.5 g (B); P0 = 0 g (calculated according to van Verseveld et al. [188]; values for strains 2 through 5 in panel ? are from that publication but modified).

10.1128/9781555818388/fig60-3_thmb.gif

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Figure 3

Theoretical exoproduct formation in batch (A) and fed batch (B) cultures. In panel A, five strains are evaluated for exoproduct formation by using batch cultures (i.e., screening stage). The same five strains are run in a carbon-limited fed-batch culture (i.e., production stage) (B). The production profiles show how the amounts of product in batch cultures at any time of sampling (without knowing the a and b values in equation 22) cannot lead to reliable predictions of exoproduct formation in fed-batch cultures. For further discussion, see text. Assumptions are that in the batch culture, growth of all five strains halted after consumption of 50 mmol of glucose because a compound other than the carbon and nitrogen source became limited. Growth in the fed-batch culture proceeded glucose limited (constant feed rate [rs] = 0.001 mol/[g · h]). Exoproduct formation is described by rp = axt + brx (21). The value of a (in grams per gram · hour) is constant; b (in grams per gram) was assumed to be described as follows: 1, b = 0.117 - 0.14μ; 2, b = 0.530 - 0.83μ; 3, b = 0.530 - 0.80μ; 4, b = 0.117 - 0.10μ; 5, b = 0.117 - 0.07μ (relations that can be obtained from, e.g., chemostat experiments). Other parameter values: V = 1 liter; Yxsm =100 g/mol; Ypsm= 60 g/mol; ms = 0.00024 mol/(g · h); x0= 0.01 g (A) or 0.5 g (B); P0 = 0 g (calculated according to van Verseveld et al. [188]; values for strains 2 through 5 in panel ? are from that publication but modified).

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